Zinc is a master-regulator of sperm function associated with binding, motility, and metabolic modulation during porcine sperm capacitation

Sperm capacitation is a post-testicular maturation step endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. Recently discovered mammalian sperm zinc signatures and their changes during sperm in vitro capacitation (IVC) warranted a more in-depth study of zinc interacting proteins (further zincoproteins). Here, we identified 1752 zincoproteins, with 102 changing significantly in abundance (P < 0.05) after IVC. These are distributed across 8 molecular functions, 16 biological processes, and 22 protein classes representing 130 pathways. Two key, paradigm-shifting observations were made: i) during sperm capacitation, molecular functions of zincoproteins are both upregulated and downregulated within several molecular function categories; and ii) Huntington’s and Parkinson’s disease pathways were the two most represented, making spermatozoon a candidate model for studying neurodegenerative diseases. These findings highlight the importance of Zn2+ homeostasis in reproduction, offering new avenues in semen processing for human-assisted reproductive therapy, identification of somatic-reproductive comorbidities, and livestock breeding.

− homeostasis resulting in substantially reduced sperm motility, swimming speed, and HCO 3 −enhanced beat frequency. A comparative study of carbonic anhydrases between human and mouse was performed 3 . The key findings were that capacitated human spermatozoa strongly depend on CA activity to support normal motility, while capacitated mouse spermatozoa do not.
Furthermore, the inhibition of CA activity in capacitated human spermatozoa increases the acrosomal exocytosis, but such an increase is not observed in capacitated mouse spermatozoa. In a recent review by Di Fiore et al. 6 , an extensive literature search was performed to summarize posttranslational modifications of human carbonic anhydrases. There is a widespread occurrence of modification events on all CAs often detected at conserved amino acids, including phosphorylation, non-enzymatic glycation, N-glycosylation, O-glycosylation, S-glutathionylation, S-nitrosylation, disulfide-formation, acetylation, ubiquitination, methylation, and GPI-anchoring. Despite the clear correlation between a specific PTM and the CA function in some cases, further dedicated studies will be required to elucidate the biological relevance of these modifications.
Calicin (CCIN) is a major basic protein of the mammalian head sperm cytoskeleton that was first reported as a component of bull perinuclear theca (PT) 7 . More attention was given to CCIN by Oko and Maravei 8 where the authors extracted PT proteins from the acrosome-free bull sperm heads. Among the extracted proteins, CCIN migrated at 60 kDa (hence the initial PT60 designation) and localized to both the subacrosomal layer and postacrosomal sheath of PT. The same authors showed that CCIN is engaged in the development of the acrosomic system early in spermiogenesis; and after the acrosome had capped the nucleus, the labeling resided in the subacrosomal layer of the nucleus 9 . Later, during the spermatid elongation, CCIN extended caudally over the nascent postacrosomal sheath. During spermiogenesis, CCIN associates with Factin with high affinity and is closely tied to the acrosomal formation 10 . For more in-depth reviews of PT formation during spermatogenesis, we recommend 11,12 . cDNA cloning of CCIN revealed that it contains a region of ~100 amino acids homologous to an extended motif shared by the kelch protein family as well as various zinc finger motifs 13 .
In the present study, we focused on the fate of CCIN, as one of the identified zincoproteins, during boar sperm capacitation. We immunolocalized CCIN in the postacrosomal region of ejaculated (non-capacitated) spermatozoa and the whole sperm head in IVC spermatozoa. We speculate that similarly as in bull spermatozoa, the CCIN is present in both the subacrosomal layer and postacrosomal sheath; however, the subacrosomal layer is inaccessible to the anti-CCIN antibody in ejaculated spermatozoa. During capacitation, a spermatozoon is subjected to biophysical and biochemical changes that prepare it for fertilization, Zn 2+ efflux being one of them 14 that might loosen up PT, making it more accessible for antibody labeling in the subacrosomal region. We also identified other known PT proteins within the zincoproteome, such as RAB2A and H2B. As a result of capacitation, these proteins are detected in higher abundances; for CCIN P=0.039, for RAB2A P=0.077 (Tab S1). The CCIN extractability increase in capacitated spermatozoa was also confirmed by WB of the alkali and SDS extracts ( fig. S10). Earlier, we reported a posterior-to-anterior plasma membrane and nuclear envelope modification wave that was reflected by incorporation of propidium iodide, a fluorescent tracer of increased membrane permeability, to the nuclei of live, capacitating spermatozoa in the same direction 15 . We have observed the same trend with the anti-CCIN antibody accessibility as a posterior-to-anterior wave of the CCIN fluorescence signal detection that is reflected as the presence of sperm populations with different fluorescence signal intensity in IVC spermatozoa ( fig. S6). It can be hypothesized that in addition to the plasma membrane and nuclear envelope modification during sperm capacitation, it is the PT loosening as a result of organizational changes of PT proteins that facilitates the PI incorporation into the nucleus. Whichever the case is, we can agree that CCIN as well as other PT proteins have a very important structural and regulatory role during sperm capacitation that has been overlooked.
The last of the three studied zincoprotein, CCDC39 is important for the proper assembly of inner dynein arms of cilia and flagella and the mutation of the CCDC39 gene is a cause of primary ciliary dyskinesia [ 16,17 , reviewed in 18 ]. The observed phenotype of men and dogs with CCDC39 mutation is microtubular disorganization that resulted in oligoasthenospermia 16,17 . In the present study, we found CCDC39 to be significantly more abundant in the capacitated sperm zincoproteome when compared to ejaculated one. We found CCDC39 to immunolocalize in the flagellum and the apical ridge of the acrosomal region in boar spermatozoa. While the localization and function of CCDC39 in the flagellum is indisputable, to our best knowledge, neither the localization nor the function of CCDC39 in the sperm head has been reported previously. We can speculate from the CCDC39 localization that it might play a role in the acrosomal integrity of the spermatozoon, and/or be a vestige of microtubule involvement in acrosomal biogenesis during spermatid differentiation. Interestingly, the acrosomal CCDC39 localization is shifted from a relatively wider area in ejaculated spermatozoa to a narrower region in IVC spermatozoa that is reflected by a decrease of fluorescence intensity signal monitored by IBFC ( fig. S11). CCDC39 in ejaculated and IVC sperm zincoproteome was below the limit of detection when we performed WB analysis (fig. S12); however, the CCDC39 abundance level was roughly the same in the whole sperm extract of ejaculated and IVC spermatozoa (Extended Data Fig. 2I). Judging from its dual localization, it seems that CCDC39 may play multiple roles in boar spermatozoa. It has been reported that CCDC39 is required for the correct assembly of inner dynein arms in the forming sperm flagellum during spermatogenesis 18 , however, its presence in the mature sperm flagellum has not been fully clarified to this day. Additional studies are required to elucidate the function of acrosomal CCDC39 in sperm capacitation.

Fig. S1.
Proteins bound to the Zn 2+ -immobilized beads abundantly, while only limited binding was observed with EDTA-stripped beads. To test the specificity of Immobilized Metal Ion Affinity chromatography, the extracts from ejaculated and IVC spermatozoa were incubated with Zn 2+ immobilized beads or EDTA-chelated beads. The Zn 2+ immobilized and EDTA-chelated beads' binding and non-binding fractions were resolved by 1-DE on 4-12 % Bis-Tris gel, stained with CBB, destained, and restained with acidic silver staining (cat# 1610449, Bio-Rad).

Fig. S2.
PANTHER molecular function analysis of identified zincoproteins. The outer circle represents high level (level 0), while sub-level (level 1) is represented by the inner circle of the respective doughnut charts. For clarity, some of the level 1 categories were removed; all categories are shown in Table S2.

Fig. S3.
PANTHER biological process analysis of identified zincoproteins. The outer circle represents high level (level 0), while sub-level (level 1) is represented by the inner circle of the respective doughnut charts. For clarity, the chart displays the first eight most abundant categories (cumulatively 92%); and some of the level 1 categories were removed as well; all categories for both levels are shown in Table S2. 0 (x-axis) and unadjusted P-value ≤ 0.05 (y-axis). Red circles represent zincoproteins above the fold change and unadjusted P-value threshold; green circles represent those above the unadjusted P-value threshold but below the fold change threshold; blue circles represent zincoproteins above the fold change but below the unadjusted P-value threshold; and gray circles represent those that are below both thresholds.

Fig. S5.
Immunolocalization of the three zincoproteins (red pseudocolor) in fixed and permeabilized spermatozoa that were significantly different between ejaculated and IVC spermatozoa. CA2 localizes in acrosomes and flagella in both ejaculated (A) and IVC spermatozoa (B); the insets show CA2 channel only. CCIN immunofluorescence signal was detected in the post-acrosomal segment of ejaculated spermatozoa (C) and the whole area of perinuclear theca (subacrosomal, equatorial & post-acrosomal) of IVC spermatozoa (D). CCDC39 localizes to the principal piece and apical ridge of sperm head in both ejaculated (E) and IVC (F) spermatozoa. Spermatozoa were co-stained for acrosomal integrity with peanut agglutinin lectin (PNA, green), and a nuclear stain DAPI (blue). All fluorescence channels are superimposed with the DIC brightfield channel. Scale bars represent 10 µm.

Fig. S6.
The remaining four replicates of capacitation-related decrease of CA2 fluorescence intensity, complementing the one shown in Fig 6A, with the corresponding descriptive statistics.

Fig. S7.
All eight replicates of CA2 WB detection from Fig 6B show a CA2 decrease in the sperm zincoproteome after in vitro capacitation. The red arrows point to the bands of actual CA2 ~ 32 kDa. Red rectangles show CA2 pulled down in the zincoproteome fractions with relative band abundances. The intensity of the CA2 bands in non-capacitated zincoproteome fractions was set to 1.00 as default and the relative intensity of the bands in capacitated zincoproteome fractions are related to their respective non-capacitated control. Membranes were stained with the CBB after the chemiluminescence detection for protein loading control (~ same protein load between noncapacitated and capacitated samples within the respective groups) and normalization purposes. The corresponding CBB stained membrane is depicted under its WB detection complement.

Fig. S8.
The other four replicates of the capacitation-related posterior-to-anterior spread of the CCIN signal (from Fig 6D) gave rise to five different CCIN localization patterns. The corresponding descriptive statistics for CCIN populations in capacitated spermatozoa are presented as well.   For densitometric quantification, the intensity of the 60 kDa band was used consistently throughout all 8 replicates. Membranes were stained with the CBB after the chemiluminescence detection for protein loading control and normalization purposes. The corresponding CBB stained membrane is depicted under its WB detection complement. It is apparent from the CBB stained membrane that the total protein load of zincoproteins is higher in replicates 5-8 when compared to 1-4.

Fig. S12.
Parallel (A) and sequential (B) isolation of perinuclear theca proteins and CCIN WB detection. Membranes were stained with the CBB after the chemiluminescence detection for protein normalization purposes. The corresponding CBB stained membrane is depicted under its WB detection complement. For the sequential isolation (B), the KCl fraction was precipitated and the whole precipitate (an equivalent of ~100 mil spermatozoa) was loaded on the gel; as opposed to 20 mil sperm equivalent in TrX-100 and NaOH fractions. It is apparent from the CBB stained membrane that the total protein load is the highest in the KCl fraction.  Membranes were stained with the CBB after the chemiluminescence detection for protein normalization purposes. The corresponding CBB stained membrane is depicted under its WB detection complement.

Fig. S15.
Gating approach -initial events collected from FlowSight data acquisition were gated for cells in focus as a function of brightfield gradient RMS, (a calculation of image crispness). Events in focus were further gated to analyze only single spermatozoa, plotted as brightfield intensity area by aspect ratio.

Fig. S16.
Secondary immunofluorescence controls. Formaldehyde fixed and Triton X-100 permeabilized spermatozoa were incubated with anti-CA2 or anti-CCIN primary antibodies, and methanol fixed/permeabilized spermatozoa were incubated with anti-CCDC39 primary antibody, respectively. Secondary incubation was void of secondary goat-anti-rabbit-FITC, DAPI, and PNA-